A large tidal wave triggered by a distant explosion threatens to drown the island of Atlantis. In the midst of an evacuation from the capital city, the Queen of Atlantis is caught by a strange, hypnotic blue light and lifted up into the "Heart of Atlantis", a powerful crystal protecting the city. The crystal consumes her and creates a dome barrier that protects the city's innermost district. She leaves behind a young daughter, Princess Kida, as the island sinks beneath the ocean.
Thousands of years later in 1914, Milo Thatch, a cartographer and linguist at the Smithsonian Institution who is marginalized for his research on Atlantis, believes that he has found The Shepherd's Journal, an ancient manuscript that contains directions to the lost island. After the museum board declines his proposal to search for the journal, a mysterious woman, Helga Sinclair, introduces Milo to Preston B. Whitmore, an eccentric millionaire. Whitmore has already funded a successful effort to retrieve the journal as repayment of a debt to Milo's grandfather, and recruits Milo to lead an expedition to Atlantis as soon as he receives it.
The expedition departs with a team of specialists led by Commander Lyle Rourke, who also led the journal recovery expedition. The crew includes Vinny Santorini, a demolitions expert; Mole Molière, a geologist; Dr. Joshua Sweet, a medical officer; Audrey Ramirez, a mechanic; Mrs. Wilhelmina Packard, a radio operator; and Cookie Farnsworth, a mess cook. And then they find Little Bear and his friends on board, as they decided to join the expedition, and even brought an old friend: Manaphy. And Emily also introduced them to some of her old friends, Wubbzy, Widget, Walden, Daizy, Gordon, Waffle, and Mr. Blik. And to our heroes surprise, Ernie is there too. He states that she won't do any thing to the others, as Peter is aware of that. They set out in the Ulysses, a massive submarine, but are soon attacked by the monstrous Leviathan, a robotic lobster-like creature that guards Atlantis' entrance. The Ulysses is destroyed, but Milo, Rourke, and part of the crew escape and make their way to an underground cavern, described in the journal as the entrance to Atlantis.
After traveling through a network of caves and a dormant volcano, the team reaches Atlantis. They are greeted by Kida who resembles a young woman and discover that the Atlantean language is the basis of many existing languages (which allows the Atlanteans to understand English). Kida enlists Milo and our heroes's aid in deciphering the Atlantean written language, long forgotten by the natives and at the same time. (But as that is explained Kida has a glimpse of Sylveon's Lightsabers.) By swimming deep within the city's submerged ruins and translating underwater murals, Milo and our heroes help Kida uncover the nature of the Heart of Atlantis: it supplies the Atlanteans with power and longevity through the crystals worn around their necks. He is surprised this is not mentioned in the journal, but upon examination realizes a page is missing.
Returning to the surface with Kida, Milo and our heroes discovers Rourke and Ernie have the missing page. Rourke, Ernie, and the crew betray Milo, and our heroes intending to bring the crystal to the surface and sell it for profit. Rourke mortally wounds the King of Atlantis while trying to extract information about the crystal's location, but finds its location for himself hidden beneath the King's throne room. The crystal detects a threat and merges with Kida. Rourke, Ernie, and the mercenaries lock Kida in a crate and prepare to leave the city. Knowing that when the crystal is gone the Atlanteans will die, Milo berates his friends for betraying their consciences and ultimately convinces them to leave Rourke and Ernie and remain in Atlantis (With our heroes help. The King explains to Milo and our heroes that the crystal has developed a consciousness – it thrives on the collective emotions of the Atlanteans and provides power, protection and longevity in return. It will find a royal host when Atlantis is in danger. He then reveals that the sinking of Atlantis was caused when he attempted to use it as a weapon of war. As he dies he gives his crystal to Milo and our heroes, telling them to save Atlantis and Kida. Encouraged by Sweet, Milo and our heroes rally the crew and the Atlanteans to stop Rourke and Ernie.
In the ensuing battle inside the volcano, Helga and the other mercenaries including Rourke are all killed. But as the ship falls, Brian and Sylveon engage in a lightsaber duel with Ernie. At first, Ernie almost wins against Sylveon but Brian cuts off his arm as Sylveon then stabs Ernie in the chest. Milo, our heroes, and the others successfully fly the crystal back to the city, as the volcano erupts. With lava flowing towards the city, Kida (in her crystal form) rises into the air and creates a protective shield. The lava breaks away harmlessly, showing a restored Atlantis, and the crystal returns Kida to Milo. The surviving crew members return to the surface and promise to keep the discovery of Atlantis a secret. Having fallen in love with Kida, Milo stays behind to help her rebuild the lost empire as Kida states that after rebuilding Atlantis, Kida decides to join the team as Sylveon decides to teach her the ways of The Force. And Manaphy decides to join Team Griffin/Eeveelution, after bonding a sister relationship with Latias. And even Gordon, Waffle, and Mr. Blik decided to join the team to explore the world.
- Peter Griffin, Vinny Griffin, Glenn Quagmire, Cleveland Brown, Joe Swanson, Little Bear, Duck, Cat, Owl, Hen, Emily, and Lucy guest star in this film.
- Ernie the Giant Chicken will work for Commander Rourke in this film.
- Manaphy returns in this film.
- This film marks it's first appearances of Wubbzy, Widget, Walden, Daizy, Gordon Quid, Waffle, and Mr. Blik.
- After this adventure, Kida, Manaphy, Gordon, Waffle, and Mr. Blik join the team.
- Atlantis sinks
- Present day/At the Museum
- Meeting Preston Whitmore/the Shepard's Journel
- Boarding the sub//reuniting with old and meeting new friends
- Meeting Mole and Sweet
- Leviathan attacks the sub
- The Quest Begins
- Atlantis!/meeting Kida
- Exploring Atlantis/the secrets of the city's powers
- Returning to the surface/Rourke and Ernie betray our heroes
- The King's Story of the Crystal
- The Battle in the volcano
- Milo kills Rourke/Duel against Ernie
- Saving Atlantis
- Atlantis has returned!/Happy Ending